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Fig. 2. Localization of MARCKS fusion proteins. (A) Cells transfected with various MARCKS fusion proteins or control cells were subjected to fractionation, and the distribution of the fusion proteins between the membrane pellet (P) or cytosolic (C) fractions was assessed by western blot analysis using an antibody against DsRed. The arrow indicates DsRed protein alone at 
30 kDa in control transfected cells. (B) The localization of each MARCKS-DsRed fusion protein was assessed by microscopic analysis of DsRed immunofluorescence. Myoblasts were plated on fibronectin for 30 minutes. Control cells were transfected with a vector expressing DsRed, and all other cells were transfected with MARCKS-DsRed fusion proteins, as indicated. The wild-type (WT) form was localized at the cell membrane and focal adhesion sites (arrow), G2A as well as G2A-mPSD forms were found mostly cytosolic with a perinuclear staining pattern notable in G2A-mPSD cells, and both PSD mutants showed punctate staining patterns. Bar, 5 µm. (C) Phosphorylation of MARCKS fusion proteins. Myoblasts expressing the vector alone (control), wild-type MARCKS (WT) or the G2A mutant (G2A) were plated on fibronectin for 1 hour. The level of MARCKS phosphorylation was measured by western blot analysis using an antibody against phosphorylated MARCKS. Endogenous phosphorylated MARCKS is indicated with an asterisk. (D) Western blot analysis using an anti-DsRed antibody showing the subcellular localization of MARCKS fusion proteins prepared from overexpressing cells plated on fibronectin for 1 hour and 4 hours, as indicated, followed by cellular fractionation. Wild-type and G2A MARCKS were found mainly in the cytosol (C) after 1 hour and in the membrane pellet (P) after 4 hours on fibronectin.
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